Intratumorally administered lactoferrin in the treatment of malignantneoplasms and other hyperproliferative diseases

ABSTRACT

The present invention relates to methods of treating a hyperproliferative disease by administering a composition of lactoferrin alone or in combination with standard anti-cancer therapies.

This application is a continuation of U.S. application Ser. No.10/435,319 filed May 9, 2003, which claims the benefit of U.S.Provisional Application No. 60/379,442 filed on May 10, 2002; U.S.Provisional Application No. 60/379,441 filed on May 10, 2002 and U.S.Provisional Application No. 60/379,474 filed on May 10, 2002, which areincorporated herein by reference in their entirety.

FIELD OF INVENTION

The present invention relates to methods of treating ahyperproliferative disease by administering a composition of lactoferrinalone or in combination with standard anti-cancer therapies. Thelactoferrin composition may be administered orally, intravenously,intratumorally, or topically.

BACKGROUND OF THE INVENTION

Currently, there are few effective options for the treatment of manycommon cancer types. The course of treatment for a given individualdepends on the diagnosis, the stage to which the disease has developed,and factors such as age, sex, and general health of the patient. Themost conventional options of cancer treatment are surgery, radiationtherapy, and chemotherapy. Surgery plays a central role in the diagnosisand treatment of cancer. Typically, a surgical approach is required forbiopsy and the removal of cancerous growth. However, if the cancer hasmetastasized and is widespread, surgery is unlikely to result in a cure,and an alternate approach must be taken. Side effects of surgery includediminished structural or organ function and increased risk of infection,bleeding, or coagulation related complications. Radiation therapy,chemotherapy, biotherapy and immunotherapy are alternatives to surgicaltreatment of cancer (Mayer, 1998; Ohara, 1998; Ho et al., 1998). Thedisadvantage of many of the alternative therapies are the side effects,which can include myelosuppression, skin irritation, difficultyswallowing, dry mouth, nausea, diarrhea, hair loss, weight loss, andloss of energy (Curran, 1998; Brizel, 1998).

Lactoferrin is a single chain metal binding glycoprotein. Many cellstypes, such as monocytes, macrophages, lymphocytes, and intestinalbrush-border cells, are known to have lactoferrin receptors. In additionto lactoferrin being an essential growth factor for both B and Tlymphocytes, lactoferrin has a wide array of functions related to hostprimary defense mechanisms. For example, lactoferrin has been reportedto activate natural killer (NK) cells, induce colony stimulatingactivity, activate polymorphonuclear neutrophils (PMN), regulategranulopoeisis, enhance antibody-dependent cell cytotoxicity, stimulatelymphokine-activated killer (LAK) cell activity, and potentiatemacrophage toxicity.

Recently, bovine lactoferrin (bLF) was used as a prophylaxis for tumorformation and/or established tumors. The present invention is the firstto use lactoferrin as a treatment, not a prophylaxis, for establishedtumors.

BRIEF SUMMARY OF THE INVENTION

The present invention is directed to a method for treating ahyperproliferative disease. The method of treatment involvesintratumoral administration of lactoferrin.

An embodiment of the present invention is a method of treating ahyperproliferative disease comprising the step of administeringintratumorally to a subject a lactoferrin composition in an amountsufficient to provide an improvement in the hyperproliferative disease.The amount of the lactoferrin composition that is administered is about0.1 μg to about 10 g per day.

The lactoferrin composition is dispersed in a pharmaceuticallyacceptable carrier. More particularly, the lactoferrin is mammalianlactoferrin, for example, human or bovine. In specific embodiments, thelactoferrin is recombinant lactoferrin.

The hyperproliferative disease is further defined as cancer, whichcomprises a neoplasm. The neoplasm is selected from the group consistingof melanoma, non-small cell lung, small-cell lung, lung hepatocarcinoma,retinoblastoma, astrocytoma, gliobastoma, leukemia, neuroblastoma,squamous cell, head, neck, gum, tongue, breast, pancreatic, prostate,renal, bone, testicular, ovarian, mesothelioma, sarcoma, cervical,gastrointestinal, lymphoma, brain, colon, and bladder.

Yet further, the hyperproliferative disease is selected from the groupconsisting of rheumatoid arthritis, inflammatory bowel disease,osteoarthritis, leiomyomas, adenomas, lipomas, hemangiomas, fibromas,vascular occlusion, restenosis, atherosclerosis, pre-neoplastic lesions,carcinoma in situ, oral hairy leukoplakia, and psoriasis.

Another embodiment is a method of treating a hyperproliferative diseasecomprising the step of supplementing a systemic and/or local immunesystem in a subject by increasing the amount of lactoferrin in thevicinity of the hyperproliferative disease. The lactoferrin isadministered intratumorally. The lactoferrin enhances, stimulates and/orup-regulates interleukin-18 and Granulocyte Macrophage ColonyStimulating Factor (GM-CSF). It is envisioned that interleukin-18stimulates the production or activity of immune cells, for example Tlymphocytes or natural killer cells and GM-CSF promotes the migrationand maturation of immune cells including dendritic and other antigenpresenting cells.

Another embodiment is a method of enhancing a local immune response inthe vicinity of a tumor following the step of administeringintratumorally to the subject a lactoferrin composition. It isenvisioned that the lactoferrin composition stimulates interleukin-18and/or GM-CSF in the site of injection, which stimulates the productionor activity of immune cells, e.g., T lymphocytes or natural killercells. T lymphocytes are selected from the group consisting of CD4+,CD8+ and CD3+ cells.

In specific embodiments, the present invention is drawn to a method oftreating a hyperproliferative disease comprising administeringintratumorally to a subject a lactoferrin composition in combinationwith chemotherapy, biotherapy, immunotherapy, surgery or radiotherapy.

The foregoing has outlined rather broadly the features and technicaladvantages of the present invention in order that the detaileddescription of the invention that follows may be better understood.Additional features and advantages of the invention will be describedhereinafter which form the subject of the claims of the invention. Itshould be appreciated by those skilled in the art that the conceptionand specific embodiment disclosed may be readily utilized as a basis formodifying or designing other structures for carrying out the samepurposes of the present invention. It should also be realized by thoseskilled in the art that such equivalent constructions do not depart fromthe spirit and scope of the invention as set forth in the appendedclaims. The novel features which are believed to be characteristic ofthe invention, both as to its organization and method of operation,together with further objects and advantages will be better understoodfrom the following description when considered in connection with theaccompanying figures. It is to be expressly understood, however, thateach of the figures is provided for the purpose of illustration anddescription only and is not intended as a definition of the limits ofthe present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the present invention, reference isnow made to the following descriptions taken in conjunction with theaccompanying drawings.

FIG. 1 shows squamous cell tumor growth with and without oral,intravenous and intratumoral administration of recombinant humanlactoferrin.

FIG. 2 shows percent tumor growth inhibition in animals receivinglactoferrin, cisplatin and lactoferrin in combination with cisplatin.

FIG. 3 shows the percent tumor growth inhibition with lactoferrin incombination with various doses of cisplatin.

FIG. 4 shows the NK activity after treatment with lactoferrin.

FIG. 5 shows squamous cell tumor growth with and without intratumoraladministration of recombinant lactoferrin once or twice a day.

DETAILED DESCRIPTION OF THE INVENTION

It is readily apparent to one skilled in the art that variousembodiments and modifications can be made to the invention disclosed inthis Application without departing from the scope and spirit of theinvention.

As used herein, the use of the word “a” or “an” when used in conjunctionwith the term “comprising” in the claims and/or the specification maymean “one,” but it is also consistent with the meaning of “one or more,”“at least one,” and “one or more than one.”

The term “hyperproliferative disease” as used herein refers to anydisease or disorder in which the cells proliferate more rapidly thannormal tissue growth. Thus, a hyperproliferating cell is a cell that isproliferating more rapidly than normal cells.

The term “parenteral administration” as used herein includes any form ofadministration in which the compound is absorbed into the subjectwithout involving absorption via the intestines. Exemplary parenteraladministrations that are used in the present invention include, but arenot limited to intramuscular, intravenous, intraperitoneal,intratumoral, intraocular, or intraarticular administration.

The term “intravenous administration” as used herein includes alltechniques to deliver a lactoferrin composition to the systemiccirculation via an intravenous injection or infusion.

The term “intratumoral administration” as used herein includes alltechniques to deliver a lactoferrin composition to the site of a tumorincluding injection, electroporation, creams, lotions or other forms ofadministration.

The term “oral administration” as used herein includes oral, buccal,enteral or intragastric administration.

The term “topical administration” as used herein includes application toa dermal, epidermal, subcutaneous or mucosal surface.

The term “pharmaceutically acceptable carrier” as used herein includesany and all solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents and the like.The use of such media and agents for pharmaceutically active substancesis well know in the art. Except insofar as any conventional media oragent is incompatible with the vectors or cells of the presentinvention, its use in therapeutic compositions is contemplated.Supplementary active ingredients also can be incorporated into thecompositions.

The term “lactoferrin” or “LF” as used herein refers to native orrecombinant lactoferrin. Native lactoferrin can be obtained bypurification from mammalian milk or colostrum or from other naturalsources. Recombinant lactoferrin (rLF) can be made by recombinantexpression or direct production in genetically altered animals, plants,fungi, bacteria, or other prokaryotic or eukaryotic species, or throughchemical synthesis.

The term “subject” as used herein, is taken to mean any mammaliansubject to which the lactoferrin composition is administered accordingto the methods described herein. In a specific embodiment, the methodsof the present invention are employed to treat a human subject. Anotherembodiment includes treating a human subject suffering from ahyperproliferative disease.

The term “therapeutically effective amount” as used herein refers to anamount that results in an improvement or remediation of the symptoms ofthe disease or condition.

The term “treating” and “treatment” as used herein refers toadministering to a subject a therapeutically effective amount of alactoferrin composition so that the subject has an improvement in thedisease. The improvement is any improvement or remediation of thesymptoms. The improvement is an observable or measurable improvement.Thus, one of skill in the art realizes that a treatment may improve thedisease condition, but may not be a complete cure for the disease.Specifically, improvements in patients with cancer may include tumorstabilization, tumor shrinkage, increased time to progression, increasedsurvival or improvements in the quality of life. Beneficial effect mayalso be reflected in an improvement of the patient's immune system asmeasured by the number and activity of circulating immune cells such asCD4+ cells, CD8+ cells, NK cells and CD40+ cells.

The term “vicinity” as used herein refers to in or around the area orsite of the tumor and/or hyperproliferative disease. For example,“vicinity of a tumor” may refer to the area in or around the tumor ormargins of the tumor. Vicinity includes the area adjacent to the tumor,the area over the tumor, the area under the tumor, the margin areaaround the tumor, or the area adjacent the tumor margin area.

A. PHARMACEUTICAL COMPOSITIONS

The lactoferrin used according to the present invention can be obtainedthrough isolation and purification from natural sources, for example,but not limited to mammalian milk. The lactoferrin is preferablymammalian lactoferrin, such as bovine or human lactoferrin. In preferredembodiments, the lactoferrin is human lactoferrin produced recombinantlyusing genetic engineering techniques well known and used in the art,such as recombinant expression or direct production in geneticallyaltered animals, plants or eukaryotes, or chemical synthesis. See, i.e.,U.S. Pat. Nos. 5,571,896; 5,571,697 and 5,571,691, which are hereinincorporated by reference.

Administration of the lactoferrin compositions according to the presentinvention will be via any common route, orally, parenterally, ortopically. Exemplary routes include, but are not limited to oral, nasal,buccal, rectal, vaginal, intramuscular, intraperitoneal, intravenous,intraarterial, intratumoral or dermal. Such compositions would normallybe administered as pharmaceutically acceptable compositions as describedherein.

The compositions of the present invention may be formulated in a neutralor salt form. Pharmaceutically-acceptable salts include the acidaddition salts (formed with the free amino groups of the protein) andwhich are formed with inorganic acids such as, for example, hydrochloricor phosphoric acids, or such organic acids as acetic, oxalic, tartaric,mandelic, and the like. Salts formed with the free carboxyl groups canalso be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, histidine, procaine and thelike.

Sterile injectable solutions are prepared by incorporating thelactoferrin in the required amount in the appropriate solvent withvarious of the other ingredients enumerated above, as required, followedby filtered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Further in accordance with the present invention, the inventivecomposition suitable for oral administration is provided in apharmaceutically acceptable carrier with or without an inert diluent.The carrier should be assimilable or edible and includes liquid,semi-solid, i.e., pastes, or solid carriers. Except insofar as anyconventional media, agent, diluent or carrier is detrimental to therecipient or to the therapeutic effectiveness of a lactoferrinpreparation contained therein, its use in an orally administrablelactoferrin for use in practicing the methods of the present inventionis appropriate. Examples of carriers or diluents include fats, oils,water, saline solutions, lipids, liposomes, resins, binders, fillers andthe like, or combinations thereof.

In accordance with the present invention, the composition is combinedwith the carrier in any convenient and practical manner, i.e., bysolution, suspension, emulsification, admixture, encapsulation,microencapsulation, absorption and the like. Such procedures are routinefor those skilled in the art.

In a specific embodiment of the present invention, the composition inpowder form is combined or mixed thoroughly with a semi-solid or solidcarrier. The mixing can be carried out in any convenient manner such asgrinding. Stabilizing agents can be also added in the mixing process inorder to protect the composition from loss of therapeutic activitythrough, i.e., denaturation in the stomach. Examples of stabilizers foruse in an orally administrable composition include buffers, antagoniststo the secretion of stomach acids, amino acids such as glycine andlysine, carbohydrates such as dextrose, mannose, galactose, fructose,lactose, sucrose, maltose, sorbitol, mannitol, etc., proteolytic enzymeinhibitors, and the like. More preferably, for an orally administeredcomposition, the stabilizer can also include antagonists to thesecretion of stomach acids.

Further, the composition for oral administration which is combined witha semi-solid or solid carrier can be further formulated into hard orsoft shell gelatin capsules, tablets, or pills. More preferably, gelatincapsules, tablets, or pills are enterically coated. Enteric coatingsprevent denaturation of the composition in the stomach or upper bowelwhere the pH is acidic. See, i.e., U.S. Pat. No. 5,629,001. Uponreaching the small intestines, the basic pH therein dissolves thecoating and permits the composition to be released and absorbed byspecialized cells, i.e., epithelial enterocytes and Peyer's patch Mcells.

In another embodiment, a powdered composition is combined with a liquidcarrier such as, i.e., water or a saline solution, with or without astabilizing agent.

A specific formulation that may be used in the present invention is asolution of lactoferrin in a hypotonic phosphate based buffer that isfree of potassium where the composition of the buffer is as follows: 6mM sodium phosphate monobasic monohydrate, 9 mM sodium phosphate dibasicheptahydrate, 50 mM sodium chloride, pH 7.0±0.1. The concentration oflactoferrin in a hypotonic buffer may range from 10 microgram/ml to 100milligram/ml. This formulation may be administered via any route ofadministration, for example, but not limited to intratumoraladministration.

Further, a composition for topical administration which is combined witha semi-solid carrier can be further formulated into a gel ointment. Apreferred carrier for the formation of a gel ointment is a gel polymer.Preferred polymers that are used to manufacture a gel composition of thepresent invention include, but are not limited to carbopol,carboxymethyl-cellulose, and pluronic polymers. Specifically, a powderedlactoferrin composition is combined with an aqueous gel containing anpolymerization agent such as Carbopol 980 at strengths between 0.5% and5% wt/volume for application to the skin for treatment ofhyperproliferative disease on or beneath the skin.

Upon formulation, solutions are administered in a manner compatible withthe dosage formulation and in such amount as is therapeuticallyeffective to result in an improvement or remediation of the symptoms.The formulations are easily administered in a variety of dosage formssuch as ingestible solutions, drug release capsules and the like. Somevariation in dosage can occur depending on the condition of the subjectbeing treated. The person responsible for administration can, in anyevent, determine the appropriate dose for the individual subject.Moreover, for human administration, preparations meet sterility, generalsafety and purity standards as required by FDA Office of Biologicsstandards.

B. TREATMENT OF HYPERPROLIFERATIVE DISEASES

In accordance with the present invention, a lactoferrin compositionprovided in any of the above-described pharmaceutical carriers isadministered to a subject suspected of or having a hyperproliferativedisease. One of skill in the art can determine the therapeuticallyeffective amount of human lactoferrin to be administered to a subjectbased upon several considerations, such absorption, metabolism, methodof delivery, age, weight, disease severity and response to the therapy.

The route of administration will vary, naturally, with the location andnature of the lesion, and include, for example intradermal, transdermal,parenteral, intravenous, intramuscular, intranasal, subcutaneous,percutaneous, intratracheal, intraperitoneal, intratumoral, perfusion,lavage, direct injection, and oral administration.

Oral administration of the lactoferrin composition includes oral,buccal, enteral or intragastric administration. It is also envisionedthat the composition may be used as a food additive. For example, thecomposition is sprinkled on food or added to a liquid prior toingestion.

Intratumoral administration of the lactoferrin composition includesintratumoral injection, electroporation, or surgical or endoscopicimplantation. Intratumoral injection, or injection into the tumorvasculature is specifically contemplated for discrete, solid, accessibletumors. Local, regional or systemic administration also may beappropriate.

The hyperproliferative disease, includes but is not limited toneoplasms. A neoplasm is an abnormal tissue growth, generally forming adistinct mass that grows by cellular proliferation more rapidly thannormal tissue growth. Neoplasms show partial or total lack of structuralorganization and functional coordination with normal tissue. These canbe broadly classified into three major types. Malignant neoplasmsarising from epithelial structures are called carcinomas, malignantneoplasms that originate from connective tissues such as muscle,cartilage, fat or bone are called sarcomas and malignant tumorsaffecting hematopoietic structures (structures pertaining to theformation of blood cells) including components of the immune system, arecalled leukemias, lymphomas and myelomas. A tumor is the neoplasticgrowth of the disease cancer. As used herein, a “neoplasm”, alsoreferred to as a “tumor”, is intended to encompass hematopoieticneoplasms as well as solid neoplasms. Examples of neoplasms include, butare not limited to melanoma, non-small cell lung, small-cell lung, lung,hepatocarcinoma, retinoblastoma, astrocytoma, gliobastoma, gum, tongue,leukemia, neuroblastoma, head, neck, breast, pancreatic, prostate,renal, bone, testicular, ovarian, mesothelioma, sarcoma, cervical,gastrointestinal, lymphoma, brain, colon, bladder, myeloma, or othermalignant or benign neoplasms.

Other hyperproliferative diseases include, but are not limited toneurofibromatosis, rheumatoid arthritis, Waginer's granulomatosis,Kawasaki's disease, lupus erathematosis, midline granuloma, inflammatorybowel disease, osteoarthritis, leiomyomas, adenomas, lipomas,hemangiomas, fibromas, vascular occlusion, restenosis, atherosclerosis,pre-neoplastic lesions, carcinoma in situ, oral hairy leukoplakia, orpsoriasis, and pre-leukemias, anemia with excess blasts, andmyelodysplastic syndrome.

Particular neoplasms of interest in the present invention include, butare not limited to hematopoietic neoplasms. For example, a hematopoieticneoplasm may include acute myelogenous leukemia, acute lymphoblasticleukemia, myelodysplastic syndrome, chronic myelomonocytic leukemia,juvenile myelomonocyte leukemia, multiple myeloma, chronic lymphocyticleukemia or other malignancy of hematologic origin.

In a preferred embodiment of the present invention, the lactoferrincompositions are administered in an effective amount to decrease,reduce, inhibit or abrogate the growth of a tumor. The amount may varyfrom about 0.1 μg to about 100 g of the lactoferrin composition.Preferably, the lactoferrin composition is orally administered in therange of 1 mg to 100 g per day, more preferably about 20 mg to about 10g per day with the most preferred dose being 4.5 g per day.Intravenously administered lactoferrin can be in the range of 0.1 μg toabout to 10 g per day, more preferably about 0.1 μg to about 1 mg withthe most preferred dose being 250 mg per day. Preferably, a lactoferrincomposition is intratumorally administered in the range of 0.1 μg to 10g per day with the most preferred dose being 100 μg per day. Topically,the amount of lactoferrin may vary from about 1 μg to about 100 g oflactoferrin. Preferably, the topical gel, solution, capsule or tabletcomprises a lactoferrin concentration of about 0.01% to about 20%. Morepreferably, the topical gel, solution, capsule or tablet may comprise alactoferrin concentration of about 1% to about 8.5%.

Treatment regimens may vary as well, and often depend on tumor type,tumor location, disease progression, and health and age of the patient.Obviously, certain types of tumor will require more aggressivetreatment, while at the same time, certain patients cannot tolerate moretaxing protocols. The clinician will be best suited to make suchdecisions based on the known efficacy and toxicity (if any) of thetherapeutic formulations.

In certain embodiments, the tumor being treated may not, at leastinitially, be resectable. Treatments with the lactoferrin compositionmay increase the resectability of the tumor due to shrinkage at themargins or by elimination of certain particularly invasive portions.Following treatments, resection may be possible. Additional treatmentssubsequent to resection will serve to eliminate microscopic residualdisease at the tumor site.

Alternatively, the present invention may be used at the time of surgery,and/or thereafter, to treat residual or metastatic disease. For example,a resected tumor bed may be injected or perfused with a formulationcomprising the lactoferrin composition. The perfusion may be continuedpost-resection, for example, by leaving a catheter implanted at the siteof the surgery. Periodic post-surgical treatment is also envisioned.

Continuous administration also may be applied where appropriate, forexample, where a tumor is excised and the tumor bed is treated toeliminate residual, microscopic disease. Delivery via syringe orcatherization is preferred. Such continuous perfusion may take place fora period from about 1-2 hours, to about 6-12 hours, to about 12-24hours, to about 1-2 days, to about 1-2 weeks or longer following theinitiation of treatment. Generally, the dose of the therapeuticcomposition via continuous perfusion will be equivalent to that given bya single or multiple injections, adjusted over a period of time duringwhich the perfusion occurs. It was further contemplated that limbperfusion may be used to administer therapeutic compositions of thepresent invention, particularly in the treatment of melanomas andsarcomas.

In specific embodiments, the lactoferrin composition is given in asingle dose or multiple doses. The single dose may be administereddaily, or multiple times a day, or multiple times a week, or monthly ormultiple times a month. In a further embodiment, the lactoferrincomposition is given in a series of doses. The series of doses may beadministered daily, or multiple times a day, weekly, or multiple times aweek, or monthly, or multiple times a month.

A further embodiment of the present invention is a method of treating ahyperproliferative disease comprising the step of supplementing amucosal immune system by increasing the amount of lactoferrin in thegastrointestinal tract. Preferably, the lactoferrin is administeredorally.

Still yet, a further embodiment is a method of enhancing a mucosalimmune response in the gastrointestinal tract in a subject comprisingthe step of administering orally to said subject a lactoferrincomposition, preferably human lactoferrin. It is envisioned thatlactoferrin stimulates interleukin-18 and GM-CSF in the gastrointestinaltract, which enhance immune cells. For example, interleukin-18 enhancesT lymphocytes or natural killer cells and GM-CSF promotes maturation andmigration of immune cells including dendritic and other antigenpresenting cells. In specific embodiments, interleukin-18 (IL-18)enhances CD4+, CD8+ and CD3+ cells. It is known by those of skill in theart that IL-18 is a Thi cytokine that acts in synergy withinterleukin-12 and interleukin-2 in the stimulation of lymphocyteIFN-gamma production. Other cytokines may also be enhanced for example,but not limited to IL-1b or, IL-12 or IFN-gamma. It is also envisionedthat lactoferrin stimulates interleukin-18 following oraladministration, which inhibits angiogenesis and thereby has activityagainst tumor cells which are dependent on neovascularization.

A further embodiment of the present invention is a method of treating ahyperproliferative disease comprising the step of supplementing thesystemic immune system by increasing the amount of lactoferrin in thesystemic circulation. Preferably, the lactoferrin composition isadministered intravenously. It is envisioned that lactoferrin stimulatesinterleukin-18 and GM-CSF in the tissue, which enhance immune cells. Forexample, interleukin-18 enhances T lymphocytes or natural killer cellsand GM-CSF promotes maturation and migration of immune cells includingdendritic and other antigen presenting cells. In specific embodiments,interleukin-18 (IL-18) enhances CD4+, CD8+ and CD3+ cells. It is knownby those of skill in the art that IL-18 is a Thi cytokine that acts insynergy with interleukin-12 and interleukin-2 in the stimulation oflymphocyte IFN-gamma production. Other cytokines may also be enhancedfor example, but not limited to IL-1b or, IL-12 or IFN-gamma. It is alsoenvisioned that lactoferrin stimulates interleukin-18 followingintravenous administration, which inhibits angiogenesis and thereby hasactivity against tumor cells which are dependent on neovascularization.

A further embodiment of the present invention is a method of treating ahyperproliferative disease comprising the step of supplementing a localor systemic immune system by increasing the amount of lactoferrin in thevicinity of the tumor. Vicinity of the tumor refers to the general areaof the tumor, for example the lactoferrin can be administered directlyinto or on the tumor, or in the general area of the tumor, but notdirectly into the tumor. The general area may include the margin area ornear or adjacent the margin area of the tumor. Preferably, thelactoferrin composition is administered intratumorally. It is envisionedthat lactoferrin stimulates interleukin-18 and GM-CSF in the localtissue, which enhances immune cells. For example, interleukin-18enhances T lymphocytes or natural killer cells and GM-CSF promotesmaturation and migration of immune cells including dendritic and otherantigen presenting cells. In specific embodiments, interleukin-18(IL-18) enhances CD4+, CD8+ and CD3+ cells. It is known by those ofskill in the art that IL-18 is a Thi cytokine that acts in synergy withinterleukin-12 and interleukin-2 in the stimulation of lymphocyteIFN-gamma production. Other cytokines may also be enhanced for example,but not limited to IL-1b or, IL-12 or IFN-gamma. It is also envisionedthat lactoferrin stimulates interleukin-18 following intratumoraladministration, which inhibits angiogenesis and thereby has activityagainst tumor cells which are dependent on neovascularization.

A further embodiment of the present invention is a method of treating ahyperproliferative disease comprising the step of supplementing a localor systemic immune system by increasing the amount of lactoferrin in theskin in the vicinity of the tumor. Preferably, the lactoferrincomposition is administered topically. As above, administration in thevicinity of the tumor includes administration near or adjacent to themargins of the tumor or directly in the margin area of the tumor. It isenvisioned that lactoferrin stimulates interleukin-18 and GM-CSF in thelocal tissue (e.g., keratinocytes), which enhances immune cells. Forexample, interleukin-18 enhances T lymphocytes or natural killer cellsand GM-CSF promotes maturation and migration of immune cells includingdendritic and other antigen presenting cells. In specific embodiments,interleukin-18 (IL-18) enhances CD4+, CD8+ and CD3+ cells. It is knownby those of skill in the art that IL-18 is a Thi cytokine that acts insynergy with interleukin-12 and interleukin-2 in the stimulation oflymphocyte IFN-gamma production. Other cytokines may also be enhancedfor example, but not limited to IL-1b or, IL-12 or IFN-gamma. It is alsoenvisioned that lactoferrin stimulates interleukin-18 followingintratumoral administration, which inhibits angiogenesis and thereby hasactivity against tumor cells which are dependent on neovascularization.

C. COMBINATION TREATMENTS

In order to increase the effectiveness of the human lactoferrincomposition of the present invention, it may be desirable to combine thecomposition of the present invention with other agents effective in thetreatment of hyperproliferative disease, such as anti-cancer agents, orwith surgery. An “anti-cancer” agent is capable of negatively affectingcancer in a subject, for example, by killing cancer cells, inducingapoptosis in cancer cells, reducing the growth rate of cancer cells,reducing the incidence or number of metastases, reducing tumor size,inhibiting tumor growth, reducing the blood supply to tumor or cancercells, promoting an immune response against cancer cells or a tumor,preventing or inhibiting the progression of cancer, or increasing thelifespan of a subject with cancer. Anti-cancer agents include biologicalagents (biotherapy), chemotherapy agents, and radiotherapy agents. Moregenerally, these other compositions would be provided in a combinedamount effective to kill or inhibit proliferation of the cell. Thisprocess may involve administering the human lactoferrin composition ofthe present invention and the agent(s) or multiple factor(s) at the sametime. This may be achieved by administering a single composition orpharmacological formulation that includes both agents, or byadministering two distinct compositions or formulations, at the sametime, or at times close enough so as to result in an overlap of thiseffect, wherein one composition includes the human lactoferrincomposition and the other includes the second agent(s).

Alternatively, the lactoferrin composition of the present invention mayprecede or follow the other anti-cancer agent treatment by intervalsranging from minutes to weeks. In embodiments where the otheranti-cancer agent and lactoferrin composition are administered orapplied separately to the cell, one would generally ensure that asignificant period of time did not expire between the time of eachdelivery, such that the agent and lactoferrin composition would still beable to exert an advantageously combined effect on the cell. In suchinstances, it is contemplated that one may contact the cellwith/administer both modalities within about 1-14 days of each otherand, more preferably, within about 12-24 hours of each other. In somesituations, it may be desirable to extend the time period for treatmentsignificantly, however, where several days (2, 3, 4, 5, 6 or 7) toseveral weeks (2, 3, 4, 5, 6, 7 or 8) lapse between the respectiveadministrations.

1. Chemotherapy

Cancer therapies also include a variety of chemical based treatments.Some examples of chemotherapeutic agents include without limitationantibiotic chemotherapeutics such as Doxorubicin, Daunorubicin,Adriamycin, Mitomycin (also known as mutamycin and/or mitomycin-C),Actinomycin D (Dactinomycin), Bleomycin, Plicomycin, plant alkaloidssuch as TAXOL® (paclitaxel), Vincristine, Vinblastine, miscellaneousagents such as platinum based agents (e.g., Cisplatin (CDDP)), etoposide(VP16), Tumor Necrosis Factor, and alkylating agents such as,Carmustine, Melphalan (also known as alkeran, L-phenylalanine mustard,phenylalanine mustard, L-PAM, or L-sarcolysin, (a phenylalaninederivative of nitrogen mustard), Cyclophosphamide, Chlorambucil,Busulfan (also known as myleran), taxane based agents (e.g., docetaxel)and Lomustine.

Some examples of other agents include, but are not limited to,Carboplatin, Procarbazine, Mechlorethamine, Irinotecan, Topotecan,Ifosfamide, Nitrosurea, Etoposide (VP16), Tamoxifen, Raloxifene,Toremifene, Idoxifene, Droloxifene, TAT-59, Zindoxifene, Trioxifene, ICI182,780, EM-800, Estrogen Receptor Binding Agents, Gemcitabine,Navelbine, Farnesyl-protein transferase inhibitors, Transplatinum,5-Fluorouracil, hydrogen peroxide, and Methotrexate, Temazolomide (anaqueous form of DTIC), Mylotarg, Dolastatin-10, Bryostatin, or anyanalog or derivative variant of the foregoing.

2. Radiotherapeutic Agents

Radiotherapeutic agents and factors include radiation and waves thatinduce DNA damage for example, γ-irradiation, X-rays, UV-irradiation,microwaves, electronic emissions, radioisotopes, and the like. Therapymay be achieved by irradiating the localized tumor site with the abovedescribed forms of radiations. It is most likely that all of thesefactors effect a broad range of damage to DNA, the precursors of DNA,the replication and repair of DNA, and the assembly and maintenance ofchromosomes.

Dosage ranges for X-rays range from daily doses of 50 to 200 roentgensfor prolonged periods of time (3 to 4 weeks), to single doses of 2000 to6000 roentgens. Dosage ranges for radioisotopes vary widely, and dependon the half-life of the isotope, the strength and type of radiationemitted, and the uptake by the neoplastic cells.

3. Surgery

Approximately 60% of persons with cancer will undergo surgery of sometype, which includes preventative, diagnostic or staging, curative andpalliative surgery. Curative surgery is a cancer treatment that may beused in conjunction with other therapies, such as the treatment of thepresent invention, chemotherapy, radiotherapy, hormonal therapy, genetherapy, immunotherapy and/or alternative therapies.

Curative surgery includes resection in which all or part of canceroustissue is physically removed, excised, and/or destroyed. Tumor resectionrefers to physical removal of at least part of a tumor. In addition totumor resection, treatment by surgery includes laser surgery,cryosurgery, electrosurgery, and miscopically controlled surgery (Mohs'surgery). It is further contemplated that the present invention may beused in conjunction with removal of superficial cancers, precancers, orincidental amounts of normal tissue.

Upon excision of part of all of cancerous cells, tissue, or tumor, acavity may be formed in the body. Treatment may be accomplished byperfusion, direct injection or local application of the area with anadditional anti-cancer therapy. Such treatment may be repeated, forexample, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. Thesetreatments may be of varying dosages as well.

4. Other Biotherapy Agents

It is contemplated that other biological agents may be used incombination with the present invention to improve the therapeuticefficacy of treatment. These additional agents include, withoutlimitation, agents that affect the upregulation of cell surfacereceptors and GAP junctions, cytostatic and differentiation agents,inhibitors of cell adhesion, agents that increase the sensitivity of thehyperproliferative cells to apoptotic inducers, or other biologicalagents, as well as biotherapy such as for example, hyperthermia.

Hyperthermia is a procedure in which a patient's tissue is exposed tohigh temperatures (up to 106° F.). External or internal heating devicesmay be involved in the application of local, regional, or whole-bodyhyperthermia. Local hyperthermia involves the application of heat to asmall area, such as a tumor. Heat may be generated externally withhigh-frequency waves targeting a tumor from a device outside the body.Internal heat may involve a sterile probe, including thin, heated wiresor hollow tubes filled with warm water, implanted microwave antennae, orradiofrequency electrodes.

A patient's organ or a limb is heated for regional therapy, which isaccomplished using devices that produce high energy, such as magnets.Alternatively, some of the patient's blood may be removed and heatedbefore being perfused into an area that will be internally heated.Whole-body heating may also be implemented in cases where cancer hasspread throughout the body. Warm-water blankets, hot wax, inductivecoils, and thermal chambers may be used for this purpose.

Hormonal therapy may also be used in conjunction with the presentinvention. The use of hormones may be employed in the treatment ofcertain cancers such as breast, prostate, ovarian, or cervical cancer tolower the level or block the effects of certain hormones such astestosterone or estrogen and this often reduces the risk of metastases.

Adjuvant therapy may also be used in conjunction with the presentinvention. The use of adjuvants or immunomodulatory agents include, butare not limited to tumor necrosis factor; interferon alpha, beta, andgamma; IL-2 and other cytokines; F42K and other cytokine analogs; orMIP-1, MIP-1beta, MCP-1, RANTES, and other chemokines.

5. Immunotherapy

Immunotherapeutics, generally, rely on the use of immune effector cellsand molecules to target and destroy cancer cells. The immune effectormay be, for example, an antibody specific for some marker on the surfaceof a tumor cell. The antibody alone may serve as an effector of therapyor it may recruit other cells to actually effect cell killing. Theantibody also may be conjugated to a drug or toxin (chemotherapeutic,radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) andserve merely as a targeting agent. Alternatively, the effector may be alymphocyte carrying a surface molecule that interacts, either directlyor indirectly, with a tumor cell target. Various effector cells includecytotoxic T cells and NK cells.

It is contemplated that vaccines that are used to treat cancer may beused in combination with the present invention to improve thetherapeutic efficacy of the treatment. Such vaccines include peptidevaccines or dendritic cell vaccines. Peptide vaccines may include anytumor-specific antigen that is recognized by cytolytic T lymphocytes.Yet further, one skilled in the art realizes that dendritic cellvaccination comprises dendritic cells that are pulsed with a peptide orantigen and the pulsed dendritic cells are administered to the patient.

Examples of tumor-specific antigens that are being used as vaccines inmelanoma include, but are not limited to gp100 or MAGE-3. These antigensare being administered as peptide vaccines and/or as dendritic cellvaccines.

D. EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Inhibition of Tumor Growth by rhLF

Human squamous cell carcinoma (O12) was used. The cells were injectedinto the right flank of athymic nude mice. rhLF was administered eitherintratumorally (49 animals, 7 doses ranging from 0.05 μg to 125 μg perdose), intravenously (7 animals, 125 μg/dose) or orally (7 animals 20mg/dose). Control animals were treated with only the vehicle; no rhLFwas administered to the control animals. rhLF was administered twice aday for either five days (intravenous group) or eight days (all othergroups) starting 11 days after inoculation with tumor cells to allowformation of established tumors.

The efficacy of treatment was evaluated by measuring the solid tumorsize during and at the end of the experiment; the body weights were alsodetermined at the time of tumor measurements. As seen in FIG. 1 andTable 1, treatment with rhLF reduced rates of tumor growth relative tothe control by 46% to 80%. In fact, oral treatment with 20 mg rhLF mostsignificantly reduced the tumor growth, by 80% compared to the control(p=0.0073).

TABLE 1 Summary of tumor growth inhibition by rhLF in O12 tumor model inmice Inhibition Relative to Placebo P value Day 19* Day 28* Day 19* Day28* Intratumoral 28% 46% 0.139 0.0263** N = 49 Intravenous 55% 65%0.0666 0.0233** N = 7 Oral 76% 80% 0.0175** 0.0073*** N = 7 *followingstart of treatment **statistically significant (p < 0.05) ***highlystatistically significant (p < 0.01)

Results from this study showed that rhLF administered by multiple routessignificantly inhibited tumor growth in a squamous cell tumor model inmice, with oral administration being the most effective. Based uponthese results, it was further contemplated that oral lactoferrin affectsthe tumor by enhancing immune cell activity.

Example 2 Evaluation of rhLF in Tumor Types

Tumor cells from a broad range of tumor types are injected into theright flank of athymic nude mice. Animals are administered either rhLF,native hLF or bovine LF orally. Control animals are treated with onlythe vehicle, no rhLF is administered to the control animals. rhLF isadministered either once or twice a day for either one, five, seven orfourteen days or eight days starting approximately eleven days afterinoculation with tumor cells to allow formation of established tumors orat such other time as is generally done with standard or publishedregimens.

The efficacy of treatment is evaluated by measuring the solid tumor sizeduring and at the end of the experiment; the body weights are alsodetermined at the time of tumor measurements. The immune response ismeasured by measuring the amount of cytokines, T-cells and NK cells incirculation and in the intestine.

Example 3 Effect of Oral Administration of rhLF and bLF

Recombinant human lactoferrin and bovine lactoferrin were orallyadministered to mice, and the production of IL-18 in the small intestinewas measured.

Mice were treated for three days daily with 65 mg/kg/day of rhLF, 300mg/kg/day of rhLF or 300 mg/kg/day of bLF. For a control, mice were onlyadministered the pharmaceutical carrier. Twenty-four hours followingadministration of the LF or control for 3 days, animals were weighed andblood and serum were collected. Serum was used for cytokine ELISAassays.

Also, at these time points, animals were sacrificed and the smallintestinal tissue was removed for further analysis. Small intestinalepithelium was homogenized using a lysis buffer consisting of PBS, 1%Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulphatecontaining 10 μg/ml PhenylMetheylsulfonyl fluoride. Homogenate wascentrifuged at 15,000 rpm for 10 minutes and the supernatant stored at−80 C till it was tested for IL-18 levels.

As seen in Table 2 and Table 3, administration of rhLF at both dosessignificantly enhanced the amounts of IL-18 in both the serum and in theintestinal extract. Bovine LF caused a lesser increase in the intestinalIL-18 levels and did not increase the serum levels of IL-18.

TABLE 2 Effect of rhLF and bLF on IL-18 levels in the gut and serumIntestinal Extract (pg) Serum (pg) Control 955 141 300 mg/kg bLF 4,515134  65 mg/kg rhLF 7,879 259 300 mg/kg rhLF 8,350 328

TABLE 3 Stimulation by rhLF and bLF of IL-18 levels in the gut and serumIntestinal Extract Serum % Increase P-value % Increase P-value IncreaseOver Control 300 mg/kg bLF 373% 0.0086 −5% 0.5411  65 mg/kg rhLF 725%0.0034 84% 0.0132 300 mg/kg rhLF 775% 0.0001 132%  0.0007 Increase OverBlf  65 mg/kg rhLF  75% 0.1490 94% 0.0366 300 mg/kg rhLF  85% 0.0617145%  0.0084

Effect of Oral rhLF on NK Activity In Vivo

Balb/c naïve mice were treated orally with rhLF or placebo once a dayfor 3 days (see Table 4).

TABLE 4 Treatment Regimen Treatment* N Dose (mg/kg) Route Schedule Group1 Placebo 6 0 — — Group 2 RhLF 7 300 mg/kg/day Oral 3 days

On day 4, mice were sacrificed and spleens were collected. NK cells wereseparated using a magnetic bead cell sorting assay (MACS anti-NK-DX5)and counted. Cells were then tested in vitro for NK-activity against YACtargets using a lactate dehydrogenase (LDH) release test.

Table 5 shows that oral rhLF treatment resulted in a significantincrease of NK activity ex-vivo against YAC-target cells (10% @ 30:1versus 2.8% of ctrl group). No significant change in NK activity wasobserved in placebo treated mice.

TABLE 5 NK activity in mice treated with oral rhLF Control Low MediumHigh 0.056 0.09 0.407 Placebo RhLF-treated Raw data Raw data E:T E:T %Cytotoxicity* E:T cell E cell % Cytotoxicity* cell E cell Increasedratio mix ctrl Final mix ctrl Final over ctrl  30:1 0.281 0.215 2.860.358 0.267 9.81  7**  15:1 0.176 0.110 2.85 0.214 0.143 4.12  1.3 7.5:10.117 0.054 2.21 0.131 0.074 0.44 0 3.7:1 0.086 0.030 0.19 0.096 0.042 00 *% Cytotoxicity = [(Effector:target cell mix − effectors cell ctrl)] −low ctrl/[(high ctrl − low ctrl)] × 100 **p < 0.05 ((2-tailed p value)

Example 4 Effect of Oral Administration of rhLF on GM-CSF In Vivo

Recombinant human lactoferrin or placebo were orally administered tomice, and the production of GM-CSF in the small intestine was measured.

Mice (5 animals per group) were treated for three days daily with 300mg/kg/day of rhLF. For a control, mice were only administered thepharmaceutical carrier. Twenty-four hours following administration ofthe LF or placebo for 3 days, animals were and the small intestinaltissue was removed for further analysis. Small intestinal epithelium washomogenized using a lysis buffer consisting of PBS, 1% Nonidet P-40,0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulphate containing 10μg/ml PhenylMetheylsulfonyl fluoride. Homogenate was centrifuged at15,000 rpm for 10 minutes and the supernatant stored at −80 C till itwas tested for GM-CSF levels using an ELISA kit.

As shown in Table 6, treatment with rhLF increased the production of akey immunostimulatory cytokine, GM-CSF, in the small intestine relativeto the placebo treated animals.

TABLE 6 Effect of rhLF on GM-CSF levels in the gut and serum Mean (SEM)in pg Increase over Placebo Placebo 6.48 (0.32) — 300 mg/kg rhLF 7.74(0.19) 19.4% (p < 0.01)

Combination of hLF with Chemotherapy

A murine squamous carcinoma cell line (SCCVII) was injected into thefloor of the mouth through the neck skin of immunocompetent C3H mice(Day 0). Five days after tumor cell implantation (Day 5), a skinincision was made in the lower neck and surgical dissection revealed theestablished tumors. Tumors were measured in three dimensions withcalipers.

Tumor-bearing mice were randomized into a control group and seven groupsreceiving rhLF and/or cisplatin. RhLF (4 mg; 200 mg/Kg) was administeredonce daily by oral gavage for 8 days on days 5 through 12. Cisplatin wasadministered as a single dose of 5 mg/Kg given intraperitoneally eitherat the start of rhLF (day 5), in the middle (day 8) or at the end (day12) of rhLF therapy. Animals were sacrificed on day 12 post-implantationand the residual tumor masses were measured and processed for lateradditional analyses.

TABLE 7 Experimental Groups Description N RhLF mg/kg Cisplatin Group APlacebo 5 0 (Placebo) 0 Group B RhLF Alone 5 200 mg/kg 0 Group C CP Day5 5 0 5 mg/kg on day 5* Group D CP Day 8 5 0 5 mg/kg on day 8* Group ECP Day 12 4 0 5 mg/kg on day 12 Group F RhLF/CP-5 5 200 mg/kg 5 mg/kg onday 5* Group G RhLF/CP-8 5 200 mg/kg 5 mg/kg on day 8* Group HRhLF/CP-12 5 200 mg/kg  5 mg/kg on day 12*

Mice treated either with rhLF alone, cisplatin alone or both agents,showed a tumor growth inhibition (TGI) relative to the placebo animals.The maximum inhibition was observed in the group receiving boththerapies (Table 7 and FIG. 2).

In all cases, the animals receiving rhLF+cisplatin showed a TGI relativeto the relevant group receiving cisplatin alone. When pooled foranalysis, animals receiving rhLF+cisplatin showed a 77% TGI relative tothe placebo animals (P<0.0001), a 66% TGI relative to rhLF alone(P<0.01) and a 63% TGI relative to cisplatin alone (P<0.01).

Cisplatin dosing immediately prior to the start of rhLF (RhLF+CP-5) orduring the period of rhLF administration (RhLF+CP-8) provided greaterincremental benefit than when cisplatin was administered followingcompletion of rhLF therapy (RhLF+CP-12). However, only the straddlingregimen (RhLF+CP-8) provided a statistically significant improvement(P<0.01) TGI of 77% over cisplatin alone (CP Day 8).

TABLE 8 Tumor Growth Inhibition (TGI) by Treatment Group Growth Relativeto Placebo* Group (SEM) TGI (%) P-value A (Placebo) 741 (79) — — B (RhLFalone)  496 (155) 33% 0.0989 C (CP Day 5)  240 (137) 68% 0.0066 D (CPDay 8)  693 (146)  6% 0.3898 E (CP Day 12)  433 (175) 42% 0.0634 F(RhLF + CP-5) 14 (5) 98% <0.0001 G (RhLF + CP-8) 159 (48) 79% 0.0001 H(RhLF + CP-12) 331 (47) 55% 0.0011 C to E (All CP) 457 (96) 38% 0.0564 Fto H (All 168 (40) 77% <0.0001 rhLF/CP) *Inhibition and 1-tailed P-valuerelative to the placebo group **Inhibition and 1-tailed P-value comparedto the respective Cisplatin groups

Dose Dependence of Combining hLF with Chemotherapy

A murine squamous carcinoma cell line (SCCVII) was injected into thefloor of the mouth through the neck skin of immunocompetent C3H mice(Day 0) as described in Example 6. On Day 5 days after initialimplantation, tumors were measured for the baseline, then treated witheither cisplatin (Day 8, i.p., 5 mg/kg) alone or cisplatin plus threedoses of oral rhLF (daily by gavage for 7-8 days on days 5 through11/12). Animals were sacrificed on Day 11/12 and tumors measured. Therewas a dose dependent inhibition of tumor growth in the animals receivingboth rhLF and cisplatin as compared to the animals receiving cisplatinalone as shown in FIG. 3.

Example 5 Combination of hLF with Docetaxel

A murine squamous carcinoma cell line (SCCVII) was injected into thefloor of the mouth through the neck skin of immunocompetent C3H mice(Day 0) as described in Example 7. On Day 5 after initial implantation,tumors were measured for the baseline, then treated with either oralplacebo alone (once daily from days 5 to 12; 6 animals), placebo anddocetaxel (i.v. bolus of 31.3 mg/kg docetaxel on Day 8; 9 animals), ordocetaxel plus oral rhLF (200 mg/kg, administered once daily by gavagefrom days 5 to 12; 9 animals). Animals were sacrificed on Day 14 andtumors measured. Docetaxel alone caused an inhibition of tumor growthrelative to placebo and the combination of rhLF and docetaxel induced afurther growth inhibition. Inhibition and p-values (1-tailed) are shownin Table 9.

TABLE 9 Tumor Growth Inhibition (TGI) by Treatment Group Growth Relativeto Placebo* Relative to Docetaxel Group (SEM) TGI (%) P-value TGI (%)P-value Placebo 5,157 — — — — (497) Docetaxel 2,103 59% <0.0001 — —(209) Docetaxel + rhLF 1,288 75% <0.0001 39% 0.0175 (286)

Combination of hLF with Radiotherapy

A murine squamous carcinoma cell line (SCCVII) was injected into thefloor of mouth through the neck skin of immunocompetent C3H mice (Day0). Five days after tumor cell implantation (Day 5), a skin incision wasmade in the lower neck and surgical dissection revealed the establishedtumors. Tumors were measured in three dimensions with calipers.

Tumor-bearing mice were randomized into six groups receiving rhLF (200mg/Kg) and/or radiotherapy as described below. RhLF (4 mg; 200 mg/kg)was administered by oral gavage once daily for 8 days on days 5 through12. Radiotherapy was administered as single dose of 2 Gray given at thebeginning (day 5) or at during (day 8) rhLF-therapy. Animals weresacrificed on day 14 post-treatment and the residual tumor masses weremeasured and processed for later additional analyses.

TABLE 10 Experimental Groups Description N RhLF mg/kg* Radiation Group APlacebo 10 0 (Placebo) None Group B RhLF Alone 10 200 mg/kg None Group CRadiation Day 5 8 0 2 Gray on day 5 Group D RhLF/Rad 5 10 200 mg/Kg 2Gray on day 5 Group E Radiation Day 8 10 0 2 Gray on day 8 Group FRhLF/Rad 8 10 200 mg/kg 2 Gray on day 8 *RhLF/placebo was administeredonce daily by oral gave from Days 5 to 12.

Mice receiving rhLF alone, radiotherapy alone, or combination therapyshowed a significant tumor growth inhibition (TGI) relative to placebotreated mice. The mice receiving both rhLF and radiation showed a modestincrease in TGI over monotherapy with rhLF (28%, P<0.05) and radiation(15%, P=0.1207).

TABLE 11 Tumor Growth Inhibition (TGI) by Treatment Group: GrowthRelative to Placebo Group (SEM) Inhibition* P-value* A (Placebo) 2348(395) — B (rhLF alone) 1074 (163) 54% 0.0040 C (Radiation Day 5)  827(105) 65% 0.0021 D (rhLF/Rad 5)  750 (125) 68% 0.0006 E (Radiation Day8)  977 (112) 58% 0.0018 F (rhLF/Rad 8)  797 (119) 66% 0.0007 C/E (BothRadiation) 911 (78) 61% <0.0001 D/F (Both rhLF/Rad) 774 (84) 67% <0.0001*Inhibition and 1-tailed P-value compared to the placebo group

Thus, lactoferrin stimulated the immune system. Still further,lactoferrin in combination with cisplatin, docetaxel and radiationresulted in inhibition of tumor growth.

Example 6 Oral Administration of hLF in Humans

Recombinant human lactoferrin was orally administered to human patientswith a range of metastatic cancer types that had failed standardchemotherapy in two different studies conducted in multiple centers infour countries (Argentina, Brazil, Chile, U.S.) RhLF was administered atdoses of 1.5 to 9 grams daily in two divided doses in cycles of 14 eachwith a 14 day gap.

Tumor size progression was monitored through CT scans and tumor markerswhere available. CT scans were performed at baseline and after each8-week period once treatment was initiated, and also compared with apre-baseline scan conducted prior to enrollment in the study. Tumormarkers are measured every 4 weeks. Blood samples were collected tomeasure subclasses of circulating lymphocytes and NK cell activity.Plasma, serum and blood cell extract samples were collected to measurecirculating IL-18, IL-1, IL-2, and IL-4, IL-5, IL-10, IL-12 and IFN-γ.

Out of nineteen evaluable patients (those with a baseline CT scan and atleast one post-treatment scan), nine patients (47%) exhibited stabledisease by the RECIST criteria at the time of the first post-treatmentscan. Patients with a broad range of tumor types showed a benefit fromlactoferrin administration.

Table 12 shows the tumor response of five individual patients withdifferent tumor types. In all cases, the percent growth of the tumorsize prior to treatment of rhLF (the relevant duration of time is shownin parentheses) and the growth of the tumor in the ensuing two timeperiods, as measured by CT, showed a diminution in their rate of tumorgrowth or an actual shrinkage.

TABLE 12 Tumor Response of Patients Receiving Oral rhLF for treatment ofMetastatic Cancer Pretreatment Post Treatment 1 Post Treatment 2 Pa- %Growth % Growth % Growth tient# Cancer (Weeks) (Weeks) (Weeks) # 204Breast 40% (8) 0% (10) 0% (6.5) # 106 Melanoma 24% (19) −18% (11) Notyet done # 104 Gastric 25% (5.5) 10% (10) −5% (7) # 102 Ovarian 30% (21)−5% (10.5) −7% (8.5) # 007 Lung 160% (5.5) 13% (7.5) 12% (8.5)

Combination Therapy with Oral hLF in Humans

Recombinant human lactoferrin is orally administered to human patientsto inhibit tumor growth either alone or in combination with standardanti-cancer regimens.

Briefly, rhLF is administered using the optimum regimen and dosesidentified in Example 10 and the standard anti-cancer regimen(s) for theselected tumor type is used as part of the combination therapy. Theroute of administration and regimen of the additional anti-cancertherapy is as approved by the FDA for that indication or as described ina peer reviewed publication.

Tumor size progression is monitored through CT scans and tumor markerswhere available. CT scans are performed at baseline and after each8-week period once treatment is initiated. Tumor markers are measuredevery 4 weeks once treatment is initiated. Blood samples are collectedto measure subclasses of circulating lymphocytes and NK cell activity.Plasma, serum and blood cell extract samples are collected to measurecirculating IL-18, IL-1, IL-2, IL-4, IL-5, IL-10, and IL-12 and IFN-γ.

Example 7 Immunostimulation Following Administration of Oral rhLF

Balb/c naïve mice were treated orally with rhLF or placebo once a dayfor 3 days. One day later (day 4), mice were sacrificed and spleenscollected. NK cells were separated using a magnetic bead cell sortingassay (MACS anti-NK-DX5) and counted. Cells were then tested in vitrofor NK-activity against YAC targets using a lactate dehydrogenase (LDH)release test.

As shown in FIG. 4, oral rhLF treatment resulted in a significantincrease of NK activity ex-vivo against YAC-target cells. At a 30:1 E:Tratio rhLF administration resulted in a 243% relative increase overplacebo-treated animals (from 2.86% to 9.81%; p<0.05).

Example 8 Effect of Intravenous Administration

Recombinant lactoferrin, bovine lactoferrin and native lactoferrin areintravenously administered to animals, preferably rats, and theproduction of IL-18, IL-1, IL-2, IL-4, IL-5, IL-10, IL-12 and IFN-gammain the plasma, serum and blood packed cells are measured.

Briefly, rats are treated for fourteen consecutive days with 0.05 μg to1000 μg per dose. For a control, rats are only administered thepharmaceutical carrier. At specific time points following administrationof the LF or control for 0 days, 2 days, 3 days, 5 days, 9 days and 14days, animals are weighed and blood and serum are collected. The levelsof CD4+, CD8+ and NK cells are counted from the blood that wascollected. Plasma, serum and an extract of the blood cells are used forcytokine ELISA assays.

Also, at 24 day time point, animals are sacrificed and tissues areremoved for further analysis. Tissues are homogenized using a lysisbuffer consisting of PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, and0.1% sodium dodecyl sulphate containing 10 μg/ml PhenylMetheylsulfonylfluoride. Homogenate is centrifuged at 15,000 rpm for 10 minutes and thesupernatant stored at −80 C till it is tested for the cytokines IL-18,IL-1, IL-2, IL-4, IL-5, IL-10, IL-12 and IFN-gamma.

Example 9 Combination Chemotherapy of Intravenously rhLF with OtherAgents

Tumor cells to be tested are injected into the right flank of athymicnude mice. Animals are administered rhLF intravenously alone and incombination with other anti-cancer regimens as described in Example 13.Control animals are treated with only the vehicle; no rhLF isadministered to the control animals. rhLF is administered using regimensidentified as being optimal in the trials described in Example 13.Anti-cancer therapy is administered using standard or publishedregimens. Therapy starts approximately 11 days after inoculation withtumor cells to allow formation of established tumors or at such othertime as is generally done with standard or published regimens.

The efficacy of individual and combination treatments are evaluated bymeasuring the solid tumor size during and at the end of the experiment;the body weights are also determined at the time of tumor measurements.

Example 10 Intravenous Administration of hLF in Humans

Recombinant lactoferrin is intravenously administered to patients toinhibit tumor growth.

Briefly, rhLF at a dose of 500 mg per day for eight days to patientssuffering from unresectable or metastatic cancer. Alternatively, rhLF isadministered for one to eight days to patients suffering from metastaticcancer in daily doses of 0.1, 1, 10, 100, and 1000 mg. The dose isadministered intravenously.

Tumor size progression is monitored through CT scans and tumor markerswhere available. CT scans are performed at baseline and after each8-week period once treatment is initiated. Tumor markers are measuredevery 4 weeks. Blood samples are collected to measure subclasses ofcirculating lymphocytes and NK cell activity. Plasma, serum and bloodcell extract samples are collected to measure circulating IL-18, IL-1,IL-2, IL-4, IL-5, IL-10, IL-12 and IFN-7.

Example 11 Combination Therapy with Intravenous hLF

Recombinant lactoferrin is intravenously administered to patients toinhibit tumor growth either alone or in combination with standardanti-cancer regimens.

Briefly, rhLF is administered using the optimum regimen and dosesidentified in Example 15 and the standard anti-cancer regimen(s) for theselected tumor type is used as part of the combination therapy. Theroute of administration and regimen of the additional anti-cancertherapy is as approved by the FDA for that indication or as described ina peer reviewed publication.

Tumor size progression is monitored through CT scans and tumor markerswhere available. CT scans are performed at baseline and after each8-week period once treatment is initiated. Tumor markers are measuredevery 4 weeks once treatment is initiated. Blood samples are collectedto measure subclasses of circulating lymphocytes and NK cell activity.Plasma, serum and blood cell extract samples are collected to measurecirculating IL-18, IL-1, IL-2, IL-4, IL-5, IL-10, IL-12 and IFN-γ.

Example 12 Activity of Intratumoral rhLF

O12 human oropharyngeal squamous cell carcinoma tumor cells wereinjected to the right flank of athymic nude mice. Recombinant humanlactoferrin and vehicle controls were dosed via intratumoral injection.Each animal was administered different concentrations of rhLF in 50 μLdoses consisting of four separate injections of approximately 12.5 μL ofthe dose, at different directions and angles (approximately S/N/E/W) toensure that the dose was distributed evenly throughout the tumor(fanning).

TABLE 13 Treatment schedule of intratumor injections of recombinanthuman lactoferrin in O12 human squamous carcinoma cell tumors in nudemice Dose of rhLF per animal in group Group Regimen 0 1 2 3 4 5 E. AOnce on day 1, kill 8 0 100 μg 250 μg 500 μg 250 μg* na days later (nudemice) C Twice/day for 8 days starting on 0  25 μg  50 μg 125 μg 250 μg 500 μg Day 11 after inoculation, kill on Day 20 (nude mice)

Table 13 shows the regimen followed for each experimental group and thedose of rhLF per injection for each animal per group. In this study,rhLF was administered directly into the tumor. Each animal was trackeddaily for tumor growth by external caliper measurements of theprotruding tumor.

Using this model, significant reduction of tumor growth was evident inboth rhLF treated groups relative to the control animals. Compared tothe median tumor size for the pooled placebo samples from groups A andC, the rates of tumor growth in animals receiving a single dose of rhLF(Group A) were reduced by 50% on day 11 after the administration of rhLF(p<0.05). The rates of tumor growth in animals dosed twice daily (GroupC) were reduced by 56% when compared to the pooled control group(p<0.01) (See FIG. 5).

Example 13 Immune Stimulation Following Intratumoral rhLF

Normal C3H/HeJ mice were implanted with one of two mouse tumorsfollowing the methodology described in Example 17. Tumors used wereSCCVII and RIF mouse tumor cell lines. Following establishment of thetumors in the mice, tumors were injected intratumorally daily for 4 dayswith 250 or 500 μg rhLF per dose or with vehicle control. Twenty fourhours following the last intratumoral injection, animals were sacrificedand the blood examined for lymphocyte populations. The number ofcirculating lymphocytes were increased by 34% to 56% relative to theplacebo treated control animals (Table 14).

TABLE 14 Increase in circulating lymphocytes following intratumoraladministration of rhLF Number of Cells CD3+ CD4+ CD8+ Placebo 2104 1800785 rhLF treated 3291 2621 1054 Increase with rhLF 56% 46% 34%

Example 14 Combination Chemotherapy of hLF with Other Agents

Tumor cells to be tested are injected into the right flank of athymicnude mice. Animals are administered rhLF intratumorally alone and incombination with other anti-cancer regimens as described in Example 1 orExample 17. Control animals are treated with only the vehicle; no rhLFis administered to the control animals. Anti-cancer therapy isadministered using standard or published regimens. Therapy startsapproximately 11 days after inoculation with tumor cells to allowformation of established tumors or at such other time as is generallydone with standard or published regimens.

The efficacy of individual and combination treatments are evaluated bymeasuring the solid tumor size during and at the end of the experiment;the body weights are also determined at the time of tumor measurements.

Example 15 Intratumoral Administration of hLF

Recombinant lactoferrin is intratumorally administered to patients toinhibit tumor growth.

Briefly, rhLF at a dose of 1000 μg per day for eight days to patientssuffering from unresectable or metastatic cancer. Alternatively, rhLF isadministered for one to eight days to patients suffering from metastaticcancer in daily doses of 10, 50, 100, 500 and 1000 μg. The dose isadministered intratumorally.

Tumor size progression is monitored through CT scans and tumor markerswhere available. CT scans are performed at baseline and after each8-week period once treatment is initiated. Tumor markers are measuredevery 4 weeks. Blood samples are collected to measure subclasses ofcirculating lymphocytes and NK cell activity. Plasma, serum and bloodcell extract samples are collected to measure circulating IL-18, IL-1,IL-2, IL-4, IL-5, IL-10, IL-12 and IFN-7.

Example 16 Combination Therapy with Intratumoral hLF

Recombinant lactoferrin is intratumorally administered to patients toinhibit tumor growth either alone or in combination with standardanti-cancer regimens.

Briefly, rhLF is administered using the optimum regimen and dosesidentified in Example 20 and the standard anti-cancer regimen(s) for theselected tumor type is used as part of the combination therapy. Theroute of administration and regimen of the additional anti-cancertherapy is as approved by the FDA for that indication or as described ina peer reviewed publication.

Tumor size progression is monitored through CT scans and tumor markerswhere available. CT scans are performed at baseline and after each8-week period once treatment is initiated. Tumor markers are measuredevery 4 weeks once treatment is initiated. Blood samples are collectedto measure subclasses of circulating lymphocytes and NK cell activity.Plasma, serum and blood cell extract samples are collected to measurecirculating IL-18, IL-1, IL-2, IL-4, IL-5, IL-10, IL-12 and IFN-γ.

Example 17 Topical Administration of hLF in Humans

Recombinant lactoferrin in a gel formulation is administered to patientsto inhibit tumor growth.

Briefly, rhLF gel at strengths of 1%, 2.5% or 8.5% is applied twice aday to a skin or subcutaneous cancerous lesion in a patient withmetastatic disease. Application of rhLF gel continues till tumorprogression.

Size progression of the metastatic disease is monitored through CT scansand tumor markers where available. CT scans are performed at baselineand after each 8-week period once treatment is initiated. Tumor markersare measured every 4 weeks. Blood samples are collected to measuresubclasses of circulating lymphocytes and NK cell activity. Plasma,serum and blood cell extract samples are collected to measurecirculating IL-18, IL-1, IL-2, IL-4, IL-5, IL-10, IL-12 and IFN-γ.

Example 18 Combination Therapy with Topical hLF

Recombinant lactoferrin in a gel formulation is administered to patientsto inhibit tumor growth either alone or in combination with standardanti-cancer regimens.

Briefly, rhLF is administered using the optimum regimen and dosesidentified in Examples 22 and the standard anti-cancer regimen(s) forthe selected tumor type is used as part of the combination therapy. Theroute of administration and regimen of the additional anti-cancertherapy is as approved by the FDA for that indication or as described ina peer reviewed publication.

Size progression of the metastatic disease is monitored through CT scansand tumor markers where available. CT scans are performed at baselineand after each 8-week period once treatment is initiated. Tumor markersare measured every 4 weeks once treatment is initiated. Blood samplesare collected to measure subclasses of circulating lymphocytes and NKcell activity. Plasma, serum and blood cell extract samples arecollected to measure circulating IL-18, IL-1, IL-2, IL-4, IL-5, IL-10,IL-12 and IFN-γ.

REFERENCES CITED

All patents and publications mentioned in the specifications areindicative of the levels of those skilled in the art to which theinvention pertains. All patents and publications are herein incorporatedby reference to the same extent as if each individual publication wasspecifically and individually indicated to be incorporated by reference.

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Although the present invention and its advantages have been described indetail, it should be understood that various changes, substitutions andalterations can be made herein without departing from the spirit andscope of the invention as defined by the appended description. Moreover,the scope of the present application is not intended to be limited tothe particular embodiments of the process, machine, manufacture,composition of matter, means, methods and steps described in thespecification. As one of ordinary skill in the art will readilyappreciate from the disclosure of the present invention, processes,machines, manufacture, compositions of matter, means, methods, or steps,presently existing or later to be developed that perform substantiallythe same function or achieve substantially the same result as thecorresponding embodiments described herein may be utilized according tothe present invention. Accordingly, the appended descriptions areintended to include within their scope such processes, machines,manufacture, compositions of matter, means, methods, or steps.

1. A method of treating an established solid neoplasm selected from thegroup consisting of Squamous cell carcinoma, Breast cancer, lung cancer,Renal cell carcinoma and combinations thereof, the method comprising thestep of administering intratumorally to a human subject a humanlactoferrin composition in an amount sufficient to reduce the rate ofgrowth or shrink the established solid neoplasm in the human subject. 2.The method of claim 1, wherein the established solid neoplasm isselected from the group consisting of lung cancer, Renal cell carcinomaand combinations thereof and wherein the method comprises the step ofadministering the lactoferrin composition as a monotherapy or incombination with a therapeutically effective surgery, radiotherapyand/or cytotoxic chemotherapy.
 3. The method of claim 2, wherein theestablished solid neoplasm is Renal cell carcinoma and the methodcomprises the step of administering the lactoferrin composition as amonotherapy.
 4. The method of claim 2, wherein the established solidneoplasm is lung cancer and the method comprises the step ofadministering the lactoferrin composition in combination with achemotherapeutic agent selected from the group consisting of cytotoxicplatinum drugs, taxanes and combinations thereof.
 5. The method of claim4, wherein the chemotherapeutic agent is selected from the groupconsisting of paclitaxel, carboplatin and combinations thereof.
 6. Themethod of claim 1, wherein the established solid neoplasm is Breastcancer and the method comprises the step of administering thelactoferrin composition as a monotherapy or in combination withGemcitabine.
 7. The method of claim 1, wherein the established solidneoplasm is Squamous cell carcinoma and the method comprises the step ofadministering the human lactoferrin composition as a monotherapy or incombination with a therapeutically effective surgery, radiotherapyand/or cytotoxic chemotherapy.
 8. The method of claim 7, wherein thehuman lactoferrin composition is administered in combination with achemotherapeutic agent selected from the group consisting of cytotoxicplatinum drugs, taxanes and combinations thereof.
 9. The method of claim8, wherein the chemotherapeutic agent is selected from the groupconsisting of docetaxel, cisplatin and combinations thereof.
 10. Themethod of claim 1, wherein the established solid neoplasm is Squamouscell carcinoma and the method comprises the step of administering thelactoferrin composition in combination with radiotherapy.
 11. The methodof claim 1, wherein the amount of the human lactoferrin composition thatis administered is about 0.1 μg to about 10 g per day.
 12. The method ofclaim 1, further comprising administering a therapeutically effectiveamount of a cytotoxic chemotherapeutic agent selected from the groupconsisting of docetaxel, paclitaxel, carboplatin, cisplatin, Gemcitabineand combinations thereof.